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Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 92 No. 8 (October 15), 1998: pp. 2750-2758 A Gln747Pro Substitution in the IIb Subunit Is Responsible for a Moderate IIb3 Deficiency in Glanzmann Thrombasthenia By Seiji Tadokoro, Yoshiaki Tomiyama, Shigenori Honda, Morio Arai, Naomasa Yamamoto, Masamichi Shiraga, Satoru Kosugi, Yuzuru Kanakura, Yoshiyuki Kurata, and Yuji Matsuzawa From The Second Department of Internal Medicine, Osaka University Medical School, Osaka, Japan; the Department of Blood Transfusion, Osaka University Hospital, Osaka, Japan; the Department of Clinical Pathology, Tokyo Medical College, Tokyo, Japan; and the Department of Cardiovascular Research, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.     ABSTRACT Abstract Introduction Methods Results Discussion References To clarify a molecular defect responsible for moderate IIb3 deficiency, we examined two unrelated patients, MT and MS, suffering from type II and type I Glanzmann thrombasthenia (GT), respectively. Sequence analysis of polymerase chain reaction (PCR) fragments derived from platelet mRNA showed a single AC substitution at nucleotide (nt) 2334 leading to a Gln747 Pro in IIb in both patients. Allele-specific restriction enzyme analysis (ASRA) of genomic DNA demonstrated that patient MT was homozygous for the Gln747Pro substitution and patient MS was compound heterozygous for this substitution and for an RNA splice mutation at the consensus sequence of the splice acceptor site of exon 18 (AGAA). Furthermore, ASRA showed that, among 17 unrelated Japanese GT patients, this Gln747Pro substitution was detected in 4 patients, including MT and MS (homozygous, 2 patients; heterozygous, 2 patients). Cotransfection of Pro747IIb and 3 constructs into 293 cells resulted in moderate reduction in the amount of IIb3 within the transfected cells as well as on the cell surface. However, Pro747IIb3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of IIb3 by the MoAb PT25-2, suggesting that the mutant IIb3 possesses the ligand-binding function. The association between the mutant proIIb and 3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747Pro substitution moderately impaired both intracellular transport of the IIb3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair IIb3 expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on IIb is responsible for moderate IIb3 deficiency. © 1998 by The American Society of Hematology.     INTRODUCTION Abstract Introduction Methods Results Discussion References INTEGRIN IIb3 (platelet GPIIb-IIIa), a calcium-dependent heterodimeric complex, is a prototype integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor and plays a crucial role in normal hemostasis and platelet aggregation.1-3 The importance of this integrin has been well documented by the clinical features of Glanzmann thrombasthenia (GT), a rare autosomal recessive bleeding disorder characterized by a quantitative or qualitative abnormality of IIb3.4 Analysis of cultured human leukemic and megakaryocytic cell lines has led to a better understanding of the key steps for IIb3 biosynthesis.5-7 The IIb subunit is synthesized as a single-chain precursor, proIIb, that associates with the 3 subunit within the endoplasmic reticulum of cells. The proIIb3 complex is then transported to the Golgi apparatus, where IIb undergoes sugar modification and endoproteolytic cleavage into heavy and light chains. After these processing events within the Golgi apparatus, the mature IIb3 complex is rapidly transported to the cell surface. Classically, GT can be divided into three subgroups according to the amount of IIb3: type I has a severe IIb3 deficiency (<5% of normal), type II has a moderate IIb3 deficiency (10% to 20% of normal), and a variant has normal to near normal levels of a dysfunctional IIb3 (50% to 100% of normal).4 To date, more than 30 mutations in either the IIb or 3 gene responsible for the thrombasthenic phenotype have been identified.8,9 However, most of the reported mutations are responsible for severe IIb3 deficiency (type I GT). Among these, the single amino acid substitutions have been especially informative in defining precise structural domains of integrins that play a role in the biosynthesis and/or function. For example, Gly242  Asp (Gly273  Asp)10 and Gly418  Asp11 in IIb have been characterized in type I GT; these were highly conserved residues adjusted to the first calcium binding domain and flanking the fourth calcium binding domain of IIb, respectively. By contrast, the molecular basis for moderate IIb3 deficiency (type II GT) remains obscure. Four mutations have been reported: Leu183  Pro12 and Arg327  His13,14 in IIb; Leu117  Trp15 and Cys374  Thr16 in 3. We have recently demonstrated that the amount of IIb is much lower than that of 3 in a number of Japanese GT patients.17 Our data suggest that the molecular defect may exist more often in the IIb gene than in the 3 gene in Japanese GT patients. In this study, we describe a new single amino acid substitution (Gln747  Pro) in IIb responsible for moderate IIb3 deficiency in 4 unrelated GT patients. Among them, patient MT (type II) was homozygous for the Gln747  Pro substitution and patient MS (type I) was compound heterozygous for this substitution and a RNA splice mutation.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Patients.   Patient MT, the product of nonconsanguineous parents, was a 40-year-old Japanese woman who had a life-long history of moderate mucocutaneous bleeding. Hematological examinations showed a prolonged bleeding time and absence of platelet aggregation in response to ADP, epinephrine, and collagen, but a normal response to ristocetin. Clot retraction was normal. She was patient no. 7 in our previous report and was classified as type II GT.17 Patient MS, the product of nonconsanguineous parents, was a 44-year-old Japanese woman who was also diagnosed as a typical case of GT. Clot retraction was slightly impaired (38%; normal values, 48% to 68%). Patients MT and MS were unrelated. Antibodies.   Rabbit polyclonal antisera specific for IIb3 and murine monoclonal antibodies (MoAbs) AP2 (IIb3-specific MoAb) were generously provided by Dr Thomas J. Kunicki (Scripps Research Institute, La Jolla, CA).18 AP3 (3-specific MoAb) was a generous gift from Dr Peter Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI).19 PAC-1 (a ligand mimetic MoAb) binds specifically to activated IIb3 and was kindly provided by Dr Sanford Shattil (Scripps Research Institute).20 PT25-2 (IIb3-specific MoAb) activates IIb3 and was a kind gift from Drs Makoto Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).21 TP80 (IIb-specific MoAb) and MOPC21 were purchased from Nichirei (Tokyo, Japan) and Sigma Chemical (St Louis, MO), respectively. Synthetic ligand.   FK633(N-(N-{4-(4-Amidinophenoxy)butyl}-a-L-aspartyl-L-valine), a peptidomimetic antagonist specific for IIb3, was generously provided by Dr Jiro Seki (Fujisawa Pharmaceutical Co, Osaka, Japan).22 Immunoblot assay and flow cytometry.   Immunoblot assay using rabbit polyclonal antisera specific for IIb3 and flow cytometric analysis using various MoAbs were performed as previously described.23,24 The amount of IIb and 3 was semiquantified by densitometry using a CS 9000 dual-wavelength flying spot scanner (Shimadzu Corp, Kyoto, Japan). Amplification and analysis of platelet RNA.   Total cellular RNA of platelets was isolated from 30 mL of whole blood and IIb or 3 mRNA was specifically amplified by reverse transcription-polymerase chain reaction (RT-PCR), as previously described.24 The primers for the amplification of IIb mRNA and conditions for RT-PCR were described elsewhere.24 The following primers were constructed based on the published sequence of 325 and used for the first-round PCR of 3 mRNA: IIIa1, 5-CGGCCCCGGCCGCTCTGGGTGACTG-3 (sense, nucleotide [nt] 15-10); IIIa2, 5-CAACTCTTCAGGGAGGTCACG-3 (antisense, nt 1147-1127); IIIa3, 5-GAGCTCATCCCAGGGACCAC-3 (sense, nt 1015-1034); and IIIa4, 5-CACTGACTCAATCTCGTCACGGC-3 (antisense, nt 1974-1952). IIIa7 and IIIa8 were described elsewhere.24 The following nested primers were used for the second-round PCR: IIIa1-Sal I, 5-CTGTCGACGCGCTGGGGGCGCTG-3 (sense, nt 8-30; mismatched sequences were underlined); IIIa2-Sph I, 5-GGGCATGCACGCACTTCCAGCTC-3 (antisense, nt 1137-1114); IIIa3-Sal I, 5-GGGTCGACAGTTGGGGTTCTGTC-3 (sense, nt 1027-1049); IIIa4-Sph I, 5-GACGCATGCTCGTCACGGCAGTAACG-3 (antisense, nt 1945-1970); IIIa7-Sal I, 5-CTAGTCGACCAATGGGCTGCTGTG-3 (sense, nt 1749-1772); and IIIa8-Sph I, 5-GGCGCATGCTGATAATGATCTGAG-3 (antisense, nt 2376-2353). Nucleotide sequences of PCR products and subcloned cDNA fragments were determined by using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Allele-specific restriction enzyme analysis (ASRA).   Amplification of the region around exon 23 of the IIb gene was performed by using primers IIbE23, 5-CAGGTCTAACTTCAGTGTGGC-3 (sense, nt 13134-13154 in the IIb gene), and IIbE24, 5-CAGGATGTAGAGCAGGTC-3 (antisense, nt 13761-13744), using 250 ng of DNA as a template.26 The first-round PCR products were reamplified using primers IIbE23 and IIbE24Pvu II, 5-CTCTCACCCTCGCAGCTCAGCT-3 (antisense, nt 13355-13334; mismatched sequences were underlined). PCR products were then digested with restriction enzyme Pvu II. For the amplification of the region around exon 18 of the IIb gene, amplified DNA fragments using primers IIbE16, 5-GAGGTCGACTTACGTCTTTTGC-3 (sense, nt 9324-9344), and IIbI18, 5-GGGTTACATTGTGACTTGGCAC-3 (antisense, nt 10048-10027), were reamplified using nested primers IIbE17A, 5-ATGCCGAGCTGCAGCTG-3 (sense, nt 9501-9517), and IIbI18. PCR products were digested with Avr II. The resulting fragments were electrophoresed in a 6% polyacrylamide gel. Construction of IIb expression vectors.   The IIb and 3 cDNA constructs were cloned into a mammalian expression vector pcDNA3 (Invitrogen Corp, San Diego, CA) and generously provided by Dr Peter Newman. To construct the expression vectors containing the 2334A (wild-type [WT]) or 2334C (Pro747) form of IIb cDNA, PCR-based cartridge mutagenesis was performed. The 1,184-bp region (nt 1988-3171) of platelet IIb cDNA from patient MS, who was heterozygous for 2334A and 2334C, was amplified by RT-PCR using primers IIb5 and IIb8. Then, second-round amplification was performed using 1 µL of the first-round PCR products as a template with nested primers IIb5A, 5-CCAGATAGGAATCGCGATG-3 (sense, nt 2185-2203), and IIb8Xba I, 5-CCTTCTAGAATAGTGTAGGCTGCACC-3 (antisense, nt 3148-3123; mismatched sequence was underlined) and Vent Polymerase (New England Biolabs, Beverly, MA). The amplified fragments were digested with Rsr II and Xba I, and the resulting 823-bp fragments (nt 2318-3140) were extracted using GeneClean II kit (Bio 101, La Jolla, CA). The 2,367-bp fragment extending from the beginning of the open reading frame to nt 2317 was obtained by digesting the full-length of IIb cDNA with HindIII and Rsr II. These two fragments were double-inserted into the pcDNA3 digested with HindIII and Xba I. Single clones that encode A or C at nt 2334 were selected by PCR followed by Pvu II digestion. The selected clones were characterized by sequence analysis to verify the absence of any other substitutions and the proper insertion of the PCR cartridge into the vector. To generate an Ala747IIb construct, we performed the site-directed mutagenesis by PCR. We synthesized mismatched sense primer IIb747Ala, 5-CCGGTCCGGGCAGAGGCCGCAGTG-3 (sense, nt 2315-2338; mismatched sequences were underlined) and performed PCR using full-length IIb cDNA as a template and primers IIb747Ala and IIb8Xba I. PCR products were digested with Rsr II and Xba I. The 823-bp fragments (nt 2318-3140) were shuttled into pcDNA3 as described above. The mutant clones were characterized by sequence analysis to verify the absence of any other substitutions and the proper insertion of the PCR cartridge into the vector. The wild-type or mutant IIb construct was cotransfected into 293 cells with wild-type 3 construct by the calcium phosphate method, as previously described.27 The cells were cultured in Dulbecco's modified medium (DME) with 10% heat-inactivated fetal calf serum (FCS). Surface labeling of the transfected cells.   Surface proteins of the transfected cells were biotinylated, and immunoprecipitation using MoAbs was performed as previously described.27 Metabolic label with [35S] methionine and pulse chase.   Metabolic labeling of transfected cell was performed 1 day after transfection, as previously described.27 The cells were incubated with 0.2 mCi/mL of [35S]-methionine for 120 minutes. For pulse chase study, the cells were incubated with 0.4 mCi/mL of [35S]-methionine for 30 minutes and the medium was then changed to DME/10% FCS with 50 µg/mL of nonradioactive methionine. Cells were equally divided into five dishes and chased after 0, 2, 4, 8, and 24 hours, respectively. Immunoprecipitation was performed as previously described.27     RESULTS Abstract Introduction Methods Results Discussion References Immunoblot analysis.   We first analyzed platelet proteins from patients MT and MS in an immunoblot assay under nonreducing (not shown) and reducing conditions (Fig 1). Various amounts of platelet proteins obtained from three normal subjects were also examined to obtain a standard curve. In patient MT, the amounts of IIb and 3 were 15% and 22% of control, respectively, whereas in patient MS, IIb and 3 were 4% and 8%, respectively. Abnormal IIb or 3, such as a premature form of IIb, was not detected under nonreducing and reducing conditions in either patient. From these data, MT was classified as type II GT and MS as type I GT. View larger version (39K): [in this window] [in a new window]   Fig 1. Immunoblot analysis of platelet proteins from patients MT and MS using anti-IIb3 antibodies. Platelet proteins from GT patients MT and MS and various amounts of control platelet proteins from three normal subjects were electrophoresed on 7.5% polyacrylamide gel under reducing conditions and transferred to a nitrocellulose membrane. IIb and 3 were detected with a 1:10,000 dilution of rabbit anti-IIb3 antibodies. The amount of proteins electrophoresed is indicated at the bottom of each line. Nucleotide sequence analysis of IIb cDNA from MT and MS.   To identify the molecular defect in patients MT and MS, platelet mRNA was isolated from these patients and normal controls. The whole coding regions of IIb and 3 cDNA were amplified by RT-PCR. Examination of nucleotide sequences of the PCR fragments using an ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA) showed a single A  C substitution at nt 2334 in IIb cDNA that leads to a Gln747  Pro substitution in exon 23 of IIb (Fig 2). Patient MT appeared homozygous for the 2334A  C substitution. The homozygosity of the substitution was confirmed by nucleotide sequence analysis of PCR fragments from genomic DNA (data not shown). No other nucleotide substitution was detected in either IIb or 3 cDNA from patient MT. Patient MS was heterozygous for the 2334A  C substitution. View larger version (17K): [in this window] [in a new window]   Fig 2. Nucleotide sequence analysis of IIb cDNA from patients MT and MS. Nucleotides of IIb cDNA from patients MT and MS and normal control were amplified by RT-PCR. The amplified fragments were directly examined using Taq DyeDeoxy Terminator Cycle Sequencing kit, and samples were run and analyzed on an ABI 373A DNA sequencer. Because patient MS was heterozygous with severe deficiency, another genetic defect in the IIb gene was sought. Electrophoretic analysis of the first round of RT-PCR fragments using primers IIb3 and IIb4 in patient MS showed that two different-sized cDNAs were amplified: an expected size (1,031 bp) and a smaller size (~900 bp) (Fig 3). Each cDNA fragment was subcloned into pUC19, and nucleotide sequences were analyzed. Sequence analysis of the smaller-sized fragment showed that a 126-bp region corresponding to the whole nucleotide sequence of exon 18 was deleted (Fig 3). No abnormality existed in the nucleotide sequence of the expected-sized fragment. The flanking region of exon 18 of the IIb gene was then amplified from genomic DNA of patient MS as well as control by PCR using primers IIbE16 and IIbI18. Nucleotide sequence showed an AG  AA substitution at the consensus splice acceptor site (1) of exon 18 (Fig 3). No other nucleotide substitution was detected in either IIb or 3 cDNA in patient MS. Thus, MS appeared to be heterozygous for the 2334A  C substitution and the G  A substitution at the splice acceptor site of exon 18 in the IIb gene. View larger version (30K): [in this window] [in a new window]   Fig 3. Analysis of IIb cDNA and the IIb gene in patient MS. (A) Amplification of IIb cDNA from patient MS by RT-PCR. Two hundred fifty nanograms of total cellular RNA from MS or a normal control was amplified by RT-PCR using primers IIb3 and IIb4. The PCR products were electrophoresed on 1.5% agarose gel. (B) Nucleotide sequence analysis of IIb cDNA from patient MS. The cDNA PCR fragments were subcloned into pUC19, and nucleotides were sequenced. (C) Nucleotide sequence analysis of the IIb gene from patient MS. Nucleotide of the IIb gene from patient MS or a normal control was amplified by PCR using primers IIbE16 and IIbI18 and sequenced. (D) Schematic diagram indicates the mechanism of exon18 skipping of the platelet IIb mRNA. ASRA.   To confirm that patient MS was a compound heterozygote, exon 23 and exon 18 with their flanking regions were amplified by PCR, followed by digestion with Pvu II and Avr II, respectively. A restriction site for Pvu II would be created by the 2334A  C substitution and a restriction site for Avr II would be abolished by the G  A substitution. ASRA clearly indicated that the A  C substitution in exon 23 was derived from the patient's father and that the G  A substitution at the splice acceptor site of exon 18 was derived from the mother (Fig 4). These data confirmed that patient MS was a compound heterozygote. ASRA further confirmed that patient MT was homozygous for the A  C substitution in exon 23 (data not shown). Using ASRA, we examined the presence of the 2334A  C substitution in 15 other unrelated Japanese GT patients (type I, 8 cases; type II, 7 cases) and 20 control subjects. This substitution was present in 2 type II GT patients who were homozygote and heterozygote, respectively (data not shown). None of control subjects had this substitution. View larger version (37K): [in this window] [in a new window]   Fig 4. ASRA. (A) The region around exon 23 of the IIb gene was amplified by PCR using primers IIbE23 and IIbE24Pvu II, followed by digestion with Pvu II. The A  C substitution creates a restriction site for Pvu II and yields 202-bp and 20-bp fragments. The resulting fragments were electrophoresed in a 6% polyacrylamide gel. (B) The region around exon 18 of the IIb gene was amplified by PCR using primers IIbE17A and IIbI18, followed by digestion with Avr II. Avr II digestion of the PCR products yields 330-bp and 218-bp fragments in the normal allele. The G  A substitution abolished a restriction site for Avr II. The resulting fragments were electrophoresed in a 1.5% agarose gel. F, M, P, and C denote DNA from the patient's father, mother, patient (MS), and control, respectively. Undigested PCR fragment from the control is also shown (U). Marker: X174 digested with Hae III. Effect of Gln747  Pro substitution on IIb3 expression.   To examine whether the 2334A  C substitution leading to Gln747  Pro substitution (Pro747) in IIb might be responsible for type II GT, we constructed an expression vector that contained the wild-type or mutant Pro747 form of IIb. Each vector was cotransfected with the wild-type 3 cDNA into 293 cells. RNA blot analysis showed that the efficiency of transfection between the wild-type and the mutant Pro747IIb was essentially the same (data not shown). Flow cytometric analysis using the IIb-specific MoAb, TP80; the 3-specific MoAb, AP3; and the IIb3 complex-specific MoAb, AP2, showed that the level of mutant Pro747IIb3 expression was moderately reduced compared with wild-type IIb3 expression (Fig 5). Immunoprecipitation of surface-labeled transfected cells using AP2 MoAb also showed that the amount of Pro747IIb3 complex was moderately reduced compared with wild-type and that the molecular weight of the mutant IIb was the same as the wild-type (Fig 6A). Interestingly, in the mutant Pro747IIb3 transfected cells, a significant amount of a premature form of IIb (proIIb) was precipitated by AP2 MoAb. These data indicate that proIIb could be expressed and complexed with 3 on the surface of the mutant Pro747IIb3 transfected cells. Densitometric analysis showed approximately 20% of normal levels of IIb (proIIb + IIb) and approximately 29% of normal levels of 3 expressed on the surface of the Pro747IIb3 transfectants (mean of 2 separate experiments). Employing immunoblot assay using polyclonal antisera specific for IIb3, we also examined the amount of IIb3 in transfected cells. Again, the mature forms of Pro747IIb and 3 in mutant transfected cells were moderately reduced compared with wild-type transfected cells (30% of normal levels of IIb, 48% of normal levels of 3, n = 2; Fig 6B). However, the amount of proIIb was not reduced in mutant transfected cells (100% of normal levels of proIIb, n = 2; Fig 6B). These data indicate that the 2334A  C substitution leads to moderate reduction in the amount of IIb3 within the transfected cells as well as on the cell surface. View larger version (23K): [in this window] [in a new window]   Fig 5. Flow cytometric analysis of IIb3 on the transfected cell surface. Recombinant IIb cDNA containing the 2334A  C substitution subcloned into pcDNA3 was cotransfected with recombinant wild-type 3 cDNA in 293 cells. The transfected cells were incubated with TP80 (IIb-specific MoAb), AP3 (3-specific MoAb), or AP2 (IIb3 complex-specific MoAb) for 30 minutes on ice and washed once, and bound antibodies were detected by FITC-conjugated goat F(ab)2 antimouse IgG. Results are expressed as histograms of cell number (linear scale) on the ordinate versus fluorescence intensity (log scale) on the abscissa. View larger version (21K): [in this window] [in a new window]   Fig 6. Expression of IIb containing the Gln747  Pro (Pro747) mutation in transfected cells. (A) Immunoprecipitation analysis of biotin surface-labeled transfected cells. Wild-type or the mutant Pro747 form of IIb cDNA was cotransfected with wild-type 3 cDNA into 293 cells. The transfected cells were surface labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP2 (IIb3 complex-specific MoAb). Precipitates were separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After transferring to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence. (B) Immunoblot analysis of transfected cells. The transfected cells were lysed and separated by 6% SDS-PAGE under reducing conditions 2 days after transfection. After transferring to a nitrocellulose membrane, IIb and 3 were detected with polyclonal anti-IIb3 antisera. Effect of Pro747 mutant on IIb3 biosynthesis.   To elucidate the mechanism of impaired expression of the mutant IIb3, we examined the association between the mutant Pro747proIIb and 3. Transfected cells were labeled with [35S]-methionine for 2 hours; immunoprecipitation using TP80 MoAb or AP3 MoAb was then performed. Densitometric analysis of the immunoprecipitate showed that the 3/(proIIb + mature IIb) ratios were essentially the same between wild-type and the mutant transfected cells. They were 0.66 (wild-type) and 0.67 (mutant) using TP80 and 1.93 (wild-type) and 1.91 (mutant) using AP3 (n = 2; Fig 7A). These results demonstrated that the association of the mutant proIIb with 3 was the same as that of wild-type proIIb. The densitometric analysis also showed that wild-type and the mutant transfected 293 cells synthesized 3 in excess compared with proIIb and that approximately 70% of labeled 3 was still in the free form. View larger version (39K): [in this window] [in a new window]   Fig 7. Effect of the Pro747 mutant on IIb3 biosynthesis. (A) Association of wild-type or the Pro747 mutant proIIb with wild-type 3. Wild-type or the Pro747 mutant IIb3 transfected cells were labeled with 0.2 mCi/mL of [35S]-methionine for 120 minutes and total cellular lysates were prepared. Subunit association was assessed by immunoprecipitation with TP80 (IIb-specific MoAb) or AP3 (3-specific MoAb). Precipitates were separated by 6% SDS-PAGE under reducing conditions. (B) Pulse chase analysis of the stability of wild-type and Pro747 mutant proIIb subunit. Wild-type or Pro747IIb cDNA was transfected into 293 cells without the wild-type 3 cDNA, and cells were labeled with 0.4 mCi/mL of [35S]-methionine for 30 minutes and chased with media containing 50 µg/mL of nonradioactive methionine for various periods of time, as indicated. Immunoprecipitation was performed using TP80. Precipitates were separated by 6% SDS-PAGE under reducing conditions. (C) Pulse chase analysis of wild-type or the Pro747 mutant IIb3 in transfected cells. Wild-type or Pro747 IIb3 transfected cells were labeled with 0.4 mCi/mL of [35S]-methionine for 30 minutes and chased with media containing 50 µg/mL of nonradioactive methionine for various periods of time, as indicated. Immunoprecipitation was performed using TP80. Precipitates were separated by 6% SDS-PAGE under reducing conditions. This figure shows a representative of six separate experiments. (D) Densitometric analysis of the kinetics of biosynthesis of IIb3 shown in (C). The bands corresponding to proIIb (), IIb (), and 3 () were analyzed by scanning densitometry. The results were normalized relative to dye-front band at each lane. The fate of the recombinant proteins was further examined in pulse-chase experiments. First, we examined the stability of the mutant Pro747proIIb. The IIb transfected cells were pulsed with [35S]-methionine for 30 minutes, chased with unlabeled methionine for various periods of time, and then immunoprecipitated using TP80 MoAb. As shown in Fig 7B, Pro747 mutation did not affect the stability of the proIIb subunit. Next, to examine the effect of this mutation on the kinetics of IIb3 complex formation, wild-type or Pro747IIb was cotransfected with wild-type 3. Pulse-chase experiments showed that the mutant proIIb was clearly detected at 24 hours postchase and was more stable than wild-type proIIb when assembled with 3. The mutant Pro747proIIb was cleaved into heavy and light chains. However, this process was moderately impaired compared with wild-type proIIb (Fig 7C and D). Expression of site-directed Ala747IIb mutant on 293 cells.   To further examine the role of the Gln residue at amino acid 747 of IIb on IIb3 expression, we introduced a Gln747  Ala mutation (Ala747) by PCR-based site-directed mutagenesis. The mutant Ala747 form of IIb cDNA was cotransfected with wild-type 3 cDNA into 293 cells. Flow cytometric analysis using AP2 MoAb showed that the level of surface expression of Ala747IIb3 complex on the transfected cells was almost the same as wild-type IIb3 complex (mean fluorescence intensity: 81.9 for wild-type and 92.3 for Ala747IIb3, n = 2; Fig 8). These data indicate that the Gln747  Ala mutation does not impair IIb3 expression. View larger version (24K): [in this window] [in a new window]   Fig 8. Effect of Gln747  Ala substitution on IIb3 expression and PAC-1 binding to wild-type and mutant IIb3 activated with PT25-2 MoAb. The IIb cDNA containing the Gln747  Ala substitution was cotransfected with wild-type 3 cDNA into 293 cells. The transfected cells were incubated with AP2 or PT25-2 for 30 minutes on ice and washed once, and bound antibodies were detected by FITC-conjugated goat F(ab)2 antimouse IgG. For PAC-1 binding, PT25-2-treated or FK633-treated 293 cells were incubated with FITC-labeled PAC-1 for 30 minutes on ice. Results are expressed as histograms of cell number (linear scale) on the ordinate versus fluorescence intensity (log scale) on the abscissa. PAC-1 binding to wild-type and mutant IIb3.   Because the mutant Pro747IIb3 receptors were expressed at substantial levels on the surface of transfected cells, we then examined the binding of the ligand-mimetic MoAb PAC-1 in the presence of the activating MoAb PT25-2. Negative control for the PAC-1 binding was obtained using FK633, a peptidomimetic antagonist specific for IIb3. As shown in Fig 8, PAC-1 could bind to both Pro747IIb3 and Ala747IIb3 in the presence of PT25-2. The PAC-1 binding to activated IIb3 was dependent on the PT25-2 binding and the PAC-1/PT25-2 binding ratios were 1.28 and 0.97 for Pro747IIb3 and Ala747IIb3, respectively, which were normalized relative to the ratio for wild-type. These data suggest that ligand binding function of Pro747IIb3 and Ala747IIb3 is not disturbed.     DISCUSSION Abstract Introduction Methods Results Discussion References In this report, we described a new point mutation (2334A  C) leading to Gln747  Pro amino acid substitution in IIb that is responsible for moderate IIb3 deficiency (type II phenotype) in 6 of the 34 possibly mutant chromosomes in 17 unrelated Japanese GT patients. In addition, a G  A mutation at the consensus sequence of the splice acceptor site of exon 18 of the IIb gene that is likely to be responsible for the exon 18 skipping in IIb cDNA was also found. The exon 18 skipping leads to an in-frame deletion of 42 amino acids in the extracellular domain of IIb. Together with the Pro747 mutation, the deletion of exon 18 contributed to the severe reduction in IIb3 expression (in type I GT patient MS). We demonstrated that the Pro747 substitution in IIb was not a naturally occurring polymorphism of IIb. ASRA showed that none of 20 control subjects possessed this substitution. Mammalian expression vectors encoding the mutant Pro747 form of IIb were constructed and cotransfected with wild-type 3 cDNA into 293 cells. Both flow cytometric and immunoprecipitation analysis using anti-IIb3 MoAbs demonstrated that the Pro747 substitution directly leads to moderate reduction in IIb3 expression on the cell surface (20% to 30% of wild-type). The impairment of reactivity with a panel of MoAbs was not due to disruption of their epitopes, because immunoblot analysis using polyclonal anti-IIb3 antisera clearly showed reduction in the total amount of IIb3 in transfected cells. Recently, it has been demonstrated that the Leu183  Pro mutation in IIb leads to both quantitative and qualitative abnormalities in IIb3.12 However, the Gln747  Pro mutation did not impair the ligand-binding function. These results demonstrate that the Pro747 mutation only leads to a quantitative abnormality. Characterization of the two mutations (Asp242 and Asp418) flanking the calcium-binding domains of IIb responsible for type I GT indicates that these relatively well-conserved flanking sequences are not essential for assembly of the IIb3 heterodimer; rather, they are critical for the proper folding of IIb3 to be transported to the Golgi apparatus, where IIb cleavage occurs.10,11 However, the Pro747 mutation was far from the calcium-binding domains. By means of protein analysis of the proteolytic fragments of isolated IIb3 heterodimers, Calvete et al28 have demonstrated that three regions of the IIb heavy chain were involved in interaction with 3: amino acids 486-553, 696-734, and 780-814. Because amino acid 747 was close to the 696-734 region, we examined whether the Pro747 mutation might impair assembly of IIb3 heterodimers. Immunoprecipitation analysis using metabolically labeled transfected cells clearly indicated that assembly of the mutant Pro747proIIb and 3 normally occurred. In contrast to the Asp242 and Asp418 mutations, pulse chase studies demonstrated that some of the mutant Pro747proIIb3 complexes underwent endoproteolytic cleavage of proIIb into heavy and light chains. These data suggest that some IIb3 heterodimers can be transported to the Golgi apparatus. Thus, the Pro747 mutation does not completely prevent intracellular transport of the heterodimers to the Golgi apparatus. Recently, Kolodziej et al29 demonstrated that the endoproteolytic cleavage of proIIb occurs on the carboxyl side of dibasic Arg858-Arg859. They also demonstrated that the failure of cleavage does not prevent expression of IIb3 on the cell surface. Immunoprecipitation using AP2 MoAb of biotin-labeled surface proteins showed that, in addition to mature IIb, a small amount of proIIb was expressed on the Pro747 mutant transfected cells. Pulse-chase studies demonstrated that the stability of the mutant Pro747proIIb was increased only when assembled with 3. These data suggest that the mutant proIIb complexed with 3 also impairs cleavage of IIb to some extent, probably due to the induction of a conformation in IIb that is less favorable to protease activity. Because the cleavage of proIIb into heavy and light chains is not critical for surface expression of IIb3,29 our data suggest that the Pro747 mutation impairs intracellular transport of IIb3 to the Golgi apparatus as well. Although our transfection experiments demonstrated that the mutation led to the expression of a significant amount of proIIb on the transfected cells, proIIb was not detected in platelets from patients MT and MS even in the immunoblot assay. This is probably due to differences between transfected cells and platelets that circulate for 10 days with no new mRNA being made. In contrast to our patients, Jung et al30 have reported a type I GT patient whose platelets contained no normal IIb, but did show a trace amount of a premature form of IIb in an immunoblot assay.30 One of the genetic defects of their patient was an RNA splicing mutation leading to skipping of exon 26 that contains the endoproteolytic cleavage site of IIb.31 However, this abnormal IIb could not be expressed on the platelet surface. Because exon 26 skipping led to loss of 42 amino acids as well as the cleavage site, it is likely that the mutation altered the conformation of IIb3 heterodimer sufficient to prevent intracellular transport in their case. We replaced Gln747 with Ala by mutagenesis to examine the role of Gln747 on IIb3 expression. In contrast to the Pro747 mutation, Ala747 substitution did not impair IIb3 expression. These data suggest that the presence of Pro747, rather than the absence of Gln747, is critical for moderately impaired IIb3 expression. Pro is an uncharged amino acid known to disrupt secondary protein structures, and a number of naturally occurring mutations creating a Pro residue leading to impaired expression or function of proteins have been reported.12,32-34 In GT patients, it has been well documented that the Ser752  Pro mutation in the cytoplasmic domain of 3 leads to a variant GT phenotype.32 Pro752 mutation makes IIb3 incapable of being activated by intracellular signals and decreases its capacity to mediate cell spreading.35,36 However, replacement of Ser752 with Ala had no adverse effects on IIb3-mediated cell spreading.37 Kahn et al38 have demonstrated that replacement of Gly242 residue with the nonpolar amino acids Ala or Val had no effect on IIb3 expression, whereas replacement with the negatively charged Glu, positively charged Lys, or nonpolar Pro caused intracellular retention of IIb3. In summary, we have described a novel point mutation, Gln747  Pro in IIb responsible for type II GT. The mutation moderately impaired the intracellular transport of IIb3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proIIb into heavy and light chains. Our in vitro studies suggest that the impairment of IIb3 is likely due to the presence of the Pro747 residue, rather than to the absence of Gln747. Molecular genetic examination of additional GT patients should provide further insight into the structural requirements for IIb3 expression, as well as differences between type I and type II GT phenotypes.     FOOTNOTES    Submitted November 11, 1997; accepted June 5, 1998.    Supported in part by grants from the Ministry of Education, Science and Culture; the Japan Society for the Promotion of Science; and the Ryoichi Naito Foundation for Medical Research.    Address reprint requests to Yoshiaki Tomiyama, MD, The Second Department of Internal Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan; e-mail: yoshi{at}hp-blood.med.osaka-u.ac.jp.    The publication costs of this article were defrayed in part by page charge payment. 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Kahn MJ, Kieber Emmons T, Vilaire G, Murali R, Poncz M, Bennett JS: Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa. Biochemistry 35:14304, 1996[Medline] [Order article via Infotrieve] © 1998 by the American Society of Hematology.   0006-4971/98/92-0029$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: G. Losonczy, N. Rosenberg, Z. Boda, G. Vereb, J. Kappelmayer, H. Hauschner, Z. Bereczky, and L. Muszbek Three novel mutations in the glycoprotein IIb gene in a patient with type II Glanzmann thrombasthenia Haematologica, May 1, 2007; 92(5): 698 - 701. [Abstract] [Full Text] [PDF] N. Rosenberg, R. Yatuv, V. Sobolev, H. Peretz, A. Zivelin, and U. 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